Horseradish peroxidase ("HRP") is a commonly used enzyme label for immunological detection systems. HRP decomposes two molecules of hydrogen peroxide, the natural substrate, into water and oxygen. HRP initiates this reaction when it donates a pair of electrons to hydrogen peroxide. The enzyme subsequently extracts electrons (oxidizes) from a suitable donor. Some electron donors become activated upon the loss of electrons and may react with each other to form a polymer which precipitates. A donor that forms an intensely colored precipitate upon release of electrons (oxidation) makes a good HRP substrate for an immunological detection system.
The low specificity of HRP for the electron donor has allowed the development of many chromogenic substrates for HRP such as 4-chloro-1-naphthol ("4CN"), 3-amino-9-ethyl-carbazole ("AEC"), and 3,3'-diaminobenzidine ("DAB") and its derivatives. However, such substrates suffer from a number of disadvantages. For example, DAB is carcinogenic and 4CN is insensitive.
Conyers et al., Anal. Biochem., 192:207-211 (1991), describes two chromogenic systems for HRP detection on membranes which use the principle of the Nadi reaction. These systems use dimethyl or diethyl analogues of p-phenylenediamine with 4-chloro-1-naphthol to generate a blue product or 3-methyl-2-benzothiazolinone hydrazone with 4-chloro-1-naphthol to generate a red product. Bos et al., J. of Immunoassay, 2(3&4):187-204 (1981), describes the use of 3,3', 5,5'-tetramethylbenzidine ("TMB") as a non-mutagenic chromogen for HRP in enzyme-immunoassays. Brand et al., BioTechniques, 8(1): 58-60 (1990), describes a comparison of the performance and stability of two precipitating substrate systems for immunoblotting, namely, TMB and DAB. De Jong et al., Histochemical Journal 17:1119-1130 (1985), describes various visualization methods for peroxidase and alkaline phosphatase activity with respect to sensitivity, stability of chromogen solutions and final precipitates, background staining, and localization properties.
U.S. Pat. No. 4,596,770, issued to Parham et al. on Jun. 24, 1986, describes the use of aqueous N-methyl pyrrolidone as a solvent for a substrate containing tetraalkyl benzidine chromogen and a peroxide in determining peroxidase enzyme activity.
U.S. Pat. No. 4,503,143, issued to Gerber et al. on Mar. 5, 1985, describes the use of TMB or its derivatives as chromogenic substances for detection of antigen or antibody in colorimetric enzyme immunoassays.
U.S. Pat. No. 4,886,760, issued to Ben-Michael on Dec. 12, 1989, describes stable chemical compositions containing chromogenic materials and peroxides which can be used in chromogenic reactions.
Ben-Michael U.S. Pat. No. 4,849,342, issued to Ben-Michael on Jul. 18, 1989, describes a method for carrying out peroxidatively active enzyme assays when the enzyme is in free solution or in cell-bound form.
Nachlas et al., J. Histochem. Cytochem., 6:445-456(1958), describes 4-amino-1-N,N-dimethylnaphthylamine useful as a reagent for the Nadi reaction which can provide a histochemical method for either cytochrome oxidase (G-Nadi) or peroxidase (M-Nadi).
European Patent Application Publication No. 271,713 published on Jun. 22, 1988 describes a method by means of which aqueous solutions containing chromogenic materials can be prepared from substantially anhydrous compositions containing all elements of the desired chromogen.